Ureylenebis methyl-phenylene-carbonyl-bis-dihydro-2-oxo-naphthoxazine disultonic acids

ABSTRACT

Ureylenebis[methyl-phenylene carbonyl]-bis-[dihydro-2-oxo-naphth-oxazine-disulfonic acids] and salts thereof useful as complement inhibitors.

BACKGROUND OF THE INVENTION

The present invention resides in the concept of certainureylenebis[methyl-phenylenecarbonyl]bis-[dihydro-2-oxo-naphth-oxazine-disulfonic acids] and saltsthereof useful as inhibitors of the complement system of warm-bloodedanimals.

The term "complement" refers to a complex group of proteins in bodyfluids that, working together with antibodies or other factors, play animportant role as mediators of immune allergic, immunochemical and/orimmunopathological reactions. The reactions in which complementparticipates takes place in blood serum or in other body fluids, andhence are considered to be humoral reactions.

With regard to human blood, there are at present more than 11 proteinsin the complement system. These complement proteins are designated bythe letter C and by number: C1, C2, C3 and so on up to C9. Thecomplement protein C1 is actually an assembly of subunits designatedC1q, C1r and C1s. The numbers assigned to the complement proteinsreflect the sequence in which they become active, with the exception ofcomplement protein C4, which reacts after C1 and before C2. Thenumerical assignments for the proteins in the complement system weremade before the reaction sequence was fully understood. A more detaileddiscussion of the complement system and its role in body processes canbe found in, for example, Bull. World Health Org., 39, 935- 938 (1968);Scientific American, 229, (No. 5), 54-66 (1973) Medical World News,October 11, 1974, pp. 53-58, 64-66; Harvey Lectures, 66, 75-104 (1972);The New England Journal of Medicine, 287, 489-495; 545-549; 592-596;642-646 (1972); The Johns Hopkins Med. J., 128, 57-74 (1971); andFederation Proceedings, 32, 134-137 (1973).

The complement system can be considered to consist of three sub-systems:(1) a recognition unit (C1q) which enables it to combine with antibodymolecules that have detected a foreign invader; (2) an activation unit(C1r, C1s, C2, C4, C3), which prepares a site on the neighboringmembrane; and (3) an attack unit (C5, C6, C7, C8 and C9) which creates a"hole" in the membrane. The membrane attack unit is non-specific; itdestroys invaders only because it is generated in their neighborhood. Inorder to minimize damage to the host's own cells, its activity must belimited in time. This limitation is accomplished partly by thespontaneous decay of activated complement and partly by interference byinhibitors and destructive enzymes. The control of complement, however,is not perfect, and there are times when damage is done to the host'scells. Immunity is therefore a double-edged sword.

Activation of the complement system also accelerates blood clotting.This action comes about by way of the complement-mediated release of aclotting factor from platelets. The biologically active complementfragments and complexes can become involved in reactions that damage thehost's cells, and these pathogenic reactions can result in thedevelopment of immune-complex diseases. For example, in some forms ofnephritis complement damages the basal membrane of the kidney, resultingin the escape of protein from the blood into the urine. The diseasedisseminated lupus erythematosus belongs in this category; its symptomsinclude nephritis, visceral lesions and skin eruptions. The treatment ofdiphtheria or tetanus with the injection of large amounts of antitoxinsometimes results in serum sickness, an immune-complex disease.Rheumatoid arthritis also involves immune complexes. Like disseminatedlupus erythematosus, it is an autoimmune disease, in which the diseasesymptoms are caused by pathological effects of the immune system in thehost's tissues. In summary, the complement system has been shown to beinvolved with inflammation, coagulation, fibrinolysis, antibody-antigenreactions and other metabolic processes.

In the presence of antibody-antigen complexes the complement proteinsare involved in a series of reactions which may lead to irreversiblemembrane damage if they occur in the vicinity of biological membranes.Thus, while complement constitutes a part of the body's defensemechanism against infection, it also results in inflammation and tissuedamage in the immunopathological process. The nature of certain of thecomplement proteins, suggestions regarding the mode of complementbinding to biological membranes and the manner in which complementeffects membrane damage are discussed in Annual Review in Biochemistry,38, 389 (1969).

A variety of substances have been disclosed as inhibiting the complementsystem, i.e., as complement inhibitors. For example, the compounds3,3'-ureylenebis-[6-(2-amino-8-hydroxy-6-sulfo-1-naphthylazo)]benzenesulfonicacid tetrasodium salt (chlorazol fast pink), heparin and a sulphateddextran have been reported to have an anticomplementary effect, BritishJournal of Experimental Pathology, 33, 327-339 (1952). The compound8,8'-[ureylene[m-phenylenecarbonylimino(4-methyl-m-phenylene)carbonylimino]]di-1,3,5-naphthalenetrisulfonicacid, hexasodium (Suramin Sodium) as a competitive inhibitor of thecomplement system, Clin. Exp. Immunol., 10, 127-138 (1972). German Pat.No. 2,254,893 or South African Pat. No. 727,923 discloses certain1-(diphenylmethyl)-4-(3-phenylallyl)piperazines useful as complementinhibitors. U.S. Pat. No. 3,897,434 discloses certainpyrazolo[1,5c]quinazolin-5(6H)-ones useful as complement inhibitors. Thecompound m-[m-(p-nitrophenylureido)phenoxypropoxy]benzamidine is alsoknown as a complement inhibitor, Immunology, 26, 819 (1974). Otherchemical compounds having complement inhibiting activity are disclosedin, for example, Journal of Medicinal Chemistry, 12, 415-419; 902-905;1049-1056 (1969); Canadian Journal of Biochemistry, 47, 547-552 (1969);The Journal of Immunology, 104, 279-288 (1970); The Journal ofImmunology, 106, 241-245 (1971) and The Journal of Immunology, 111,1061-1066 (1973).

It has been reported that the known complement inhibitorsepsilon-aminocapronic acid, Suramin Sodium and tranexamic acid have beenused with success in the treatment of hereditary angioneurotic edema, adisease state resulting from an inherited deficiency or lack of functionof the serum inhibitor of the activated first compound of complement (C1inhibitor), The New England Journal of Medicine, 286, 808-812 (1972);Allergol, Et. Immunopath, II, 163-168 (1974); and J. Allergy Clin.Immunol., 53, No. 5, 298-302 (1974).

SUMMARY OF THE INVENTION

This invention is concerned with certain ureylenebis[methyl-phenylenecarbonyl]bis-[dihydro-2-oxo-naphth-oxazine-disulfonic acids] and salts,and their use, which can be represented by general formula (I): ##STR1##wherein A is hydrogen, alkali metal or alkaline earth, with the provisothat A is identical in the same compound. Preferably, A is sodium orpotassium.

A compound of particular interest within formula (I) is the compound3,3'-Ureylenebis[4-methyl-1,3-phenylene carbonyl]-bis[2,3-dihydro-2-oxo-naphth[1,8-de]-1,3-oxazine-5,8-disulfonic acid]tetrasodium salt which can be represented by the formula (II): ##STR2##

It has now been discovered that a representative compound encompassedwithin formulae (I) and (II) interacts with the complement reactionsequence, thereby inhibiting complement activity in body fluids.

This invention is also concerned with a method of inhibiting thecomplement system in a body fluid, such as blood serum, which comprisessubjecting body fluid complement to the action of an effectivecomplement inhibiting amount of a compound encompassed within formulae(I) and (II) hereinabove. The method of use aspect of this invention isalso concerned with a method of inhibiting the complement system in awarm-blooded animal which comprises internally administering to saidanimal an effective inhibiting amount of a compound encompassed withformula (I) and (II) hereinabove. Body fluid can include blood, plasma,serum, synovial fluid, cerebrospinal fluid, or pathologicalaccumulations of fluid as pleural effusion, etc.

The compounds of the present invention find utility as complementinhibitors in body fluids and as such may be used to ameliorate orprevent those pathological reactions requiring the function ofcomplement and in the therapeutic treatment of warm-blooded animalshaving immunologic diseases such as rheumatoid arthritis, systemic lupuserythematosus, certain kinds of glomerulonephritis, certain kinds ofauto-allergic hemolytic anemia, certain kinds of platelet disorders andcertain kinds of vasculitis. The compounds herein may also be used inthe therapeutic treatment of warm-blooded animals having non-immunologicdiseases such as paroxysmal nocturnal hemoflobinuria, hereditaryangioneurotic edema (treated with Suramin, etc.) and inflammatory statesinduced by the action of bacterial or lysosomal enzymes on theappropriate complement components as for example, inflammation followingcoronary occlusion. They may also be useful in the treatment oftransplant rejection and as blood culture on transport mediums.

The compounds of the invention may be prepared by reacting theappropriate 4-hydroxy-5-(nitro-toluamido)-naphthalenedisulfonic acid,di-salt, as illustrated in Example 1, with hydrogen in the presence ofpalladium as charcoal to obtain the corresponding4-(amino-toluamido)-5-hydroxy-naphthalenedisulfonic acid, di-salt, whichin turn is treated as illustrated in Example 2, to obtain thecorresponding ureylenebis[methyl-phenylenecarbonyl]-bis-[dihydro-2-oxo-naphth-oxazine-disulfonic] tetra-salt.Acidification produces the free acid. The closest known reference to thecompounds of the invention is J. Chem. Soc., 3068 (1927).

DETAILED DESCRIPTION OF THE INVENTION

The following examples will serve to illustrate the invention in moredetail.

EXAMPLE 1 4-(5-Amino-o-toluamido)-5-hydroxy-2,7-naphthalenedisulfonicacid, disodium salt

A mixture of 25.0 g of4-hydroxy-5-(5-nitro-o-toluamido)-2,7-naphthalenedisulfonic acid,disodium salt, 200 ml of distilled water and 2.5 g of 10% palladium oncharcoal is hydrogenated in a Parr shaker for 5 hours at roomtemperature during which time 12 pounds of hydrogen is absorbed. Themixture is heated on a steam bath and is filtered through diatomaceousearth to remove the catalyst. The filter is washed with hot water andthe filtrate is then evaporated to about 100 ml of in vacuo at 55°-60° Cwith formulation of crystals after standing at room temperatureovernight. The product of the example is collected by filtration and iswashed with absolute ethyl alcohol followed by ether then is oven driedat 120° C.

EXAMPLE 23,3'-{Ureylenebis[(6-methyl-3,1-phenylene)carbonyl]}-bis[2,3-dihydro-2-oxonaphth[1,8-de]-1,3-oxazine-5,8-disulfonicacid], tetrasodium salt

To a stirred solution of 10.0 g of4-(5-amino-o-toluamido)-5-hydroxy-2,7-naphthalenedisulfonic acid,disodium salt (prepared as described in Example 1) and 21.4 g ofanhydrous sodium carbonate in 250 ml of water is bubbled in phosgene for1 hour at room temperature. Then an additional 21.4 g of sodiumcarbonate is added and phosgenation is continued for 1 hour and 25minutes. The resulting mixture is neutralized with 16.5 g of sodiumcarbonate and is filtered and the filtrate is set aside. The precipitateis washed with a small amount of water then is oven dried at 120° C forseveral hours to give the product of the example.

                  Example 3                                                       ______________________________________                                        Preparation of Compressed Tablet                                              ______________________________________                                         Ingredient                mg/Tablet                                          ______________________________________                                        Active Compound            0.5-500                                            Dibasic Calcium Phosphate NF                                                                             qs                                                 Starch USP                 40                                                 Modified Starch            10                                                 Magnesium Stearate USP     1-5                                                ______________________________________                                    

                  Example 4                                                       ______________________________________                                        Preparation of Compressed Tablet-Sustained Action                             ______________________________________                                         Ingredient            mg/Tablet                                              ______________________________________                                        Active Compound        0.5-500 (as acid                                       as Aluminum Lake*, Micronized                                                                        equivalent)                                            Dibasic Calcium Phosphate NF                                                                         qs                                                     Alginic Acid           20                                                     Starch USP             35                                                     Magnesium Stearate USP 1-10                                                   ______________________________________                                         *Complement inhibitor plus aluminum sulfate yields aluminum complement        inhibitor. Complement inhibitor content in aluminum lake ranges from          5-30%.                                                                   

                  Example 5                                                       ______________________________________                                        Preparation of Hard Shell Capsule                                             ______________________________________                                         Ingredient                mg/Capsule                                         ______________________________________                                        Active Compound            0.5-500                                            Lactose, Spray Dried       qs                                                 Magnesium Stearate         1-10                                               ______________________________________                                    

                  Example 6                                                       ______________________________________                                        Preparation of Oral Liquid (Syrup)                                            ______________________________________                                         Ingredient                 % W/V                                             ______________________________________                                        Active Compound             0.05-5                                            Liquid Sugar                75.0                                              Methyl Paraben USP          0.18                                              Propyl Paraben USP          0.02                                              Flavoring Agent             qs                                                Purified Water qs ad        100.0                                             ______________________________________                                    

                  Example 7                                                       ______________________________________                                        Preparation of Oral Liquid (Elixir)                                           ______________________________________                                         Ingredient                 % W/V                                             ______________________________________                                        Active Compound             0.05-5                                            Alcohol USP                 12.5                                              Glycerin USP                45.0                                              Syrup USP                   20.0                                              Flavoring Agent             qs                                                Purified Water qs ad        100.0                                             ______________________________________                                    

                  Example 8                                                       ______________________________________                                        Preparation of Oral Suspension (Syrup)                                        ______________________________________                                        Ingredient                  % W/V                                             ______________________________________                                        Active Compound             0.05-5                                            as Aluminum Lake, Micronized (acid equivalent)                                Polysorbate 80 USP          0.1                                               Magnesium Aluminum Silicate,                                                  Colloidal                   0.3                                               Flavoring Agent             qs                                                Methyl Paraben USP          0.18                                              Propyl Paraben USP          0.02                                              Liquid Sugar                75.0                                              Purified Water qs ad        100.0                                             ______________________________________                                    

                  Example 9                                                       ______________________________________                                        Preparation of Injectable Solution                                            ______________________________________                                         Ingredient                 % W/V                                             ______________________________________                                        Active Compound             0.05-5                                            Benzyl Alcohol NF           0.9                                               Water for Injection         100.0                                             ______________________________________                                    

                  Example 10                                                      ______________________________________                                        Preparation of Injectable Oil                                                 ______________________________________                                         Ingredient                 % W/V                                             ______________________________________                                        Active Compound             0.05-5                                            Benzyl Alcohol              1.5                                               Sesame Oil qs ad            100.0                                             ______________________________________                                    

                  Example 11                                                      ______________________________________                                        Preparation of Intra-Articular Product                                        ______________________________________                                         Ingredient                Amount                                             ______________________________________                                        Active Compound            2-20 mg                                            NaCl (physiological saline)                                                                              0.9%                                               Sodium Carboxymethylcellulose                                                                            1-5%                                               pH adjusted to 5.0-7.5                                                        Water for Injection qs to  100%                                               ______________________________________                                    

                  Example 12                                                      ______________________________________                                        Preparation of Injectable Depo Suspension                                     ______________________________________                                         Ingredient            % W/V                                                  ______________________________________                                        Active Compound        0.05-5                                                                        (acid equivalent)                                      Polysorbate 80 USP     0.2                                                    Polyethylene Glycol 4000 USP                                                                         3.0                                                    Sodium Chloride USP    0.8                                                    Benzyl Alcohol NE      0.9                                                    HCl to pH 6-8          qs                                                     Water for Injection qs ad                                                                            100.0                                                  ______________________________________                                    

The compounds of this invention may be administered internally, e.g.,orally, or parenterally, e.g., intra-articularly, to a warm-bloodedanimal to inhibit complement in the body fluid of the animal, suchinhibition being useful in the amelioration or prevention of thosereactions dependent upon the function of complement, such asinflammatory process and cell membrane damage induced byantigen-antibody complexes. A range of doses may be employed dependingon the mode of administration, the condition being treated and theparticular compound being used. For example, for intravenous orsubcutaneous use from about 5 to about 50 mg/kg/day, or every 6 hoursfor more rapidly excreted salts, may be used. For intra-articular usefor large joints such as the knee, from about 2 to about 20 mg/joint perweek may be used, with proportionally smaller doses for smaller joints.The dosage range is to be adjusted to provide optimum therapeuticresponse in the warm-blooded animal being treated. In general, theamount of compound administered can vary over a wide range to providefrom about 5 mg/kg to about 100 mg/kg of body weight of animal per day.The usual daily dosage for a 70 kg subject may vary from about 350 mg toabout 3.5 g. Unit doses of the acid or salt can contain from about 0.5mg to about 500 mg.

In therapeutic use the compounds of this invention may be administeredin the form of conventional pharmaceutical compositions. Suchcompositions may be formulated so as to be suitable for oral orparenteral administration. The active ingredient may be combined inadmixture with a pharmaceutically acceptable carrier, which carrier maytake a wide variety of forms depending on the form of preparationdesired for administration, i.e., oral or parenteral. The compounds canbe used in compositions such as tablets. Here, the principal activeingredient is mixed with conventional tabletting ingredients such ascorn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesiumstearate, discalcium phosphate, gums, or similar materials as non-toxicpharmaceutically acceptable diluents or carriers. The tablets or pillsof the novel compositions can be laminated or otherwise compounded toprovide a dosage form affording the advantage of prolonged or delayedaction or predetermined successive action to the enclosed medication.For example, the tablet or pill can comprise an inner dosage and anouter dosage component, the latter being in the form of an envelope overthe former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permits theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids ormixtures of polymeric acids with such materials as shellac, shellac andcetyl alcohol, cellulose acetate and the like. A particularlyadvantageous enteric coating comprises a styrene maleic acid copolymertogether with known materials contributing to the enteric properties ofthe coating. The tablet or pill may be colored through the use of anappropriate non-toxic dye, so as to provide a pleasing appearance.

The liquid forms in which the novel compositions of the presentinvention may be incorporated for administration include suitableflavored emulsions with edible oils, such as, cottonseed oil, sesameoil, coconut oil, peanut oil, and the like, as well as elixirs andsimilar pharmaceutical vehicles. Sterile suspensions or solutions can beprepared for parenteral use. Isotonic preparations containing suitablepreservatives are also desirable for injection use.

The term dosage form as described herein refers to physically discreteunits suitable as unitary dosage for warm-blooded animals subjects, eachunit containing a predetermined quantity of active component calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical diluent carrier or vehicle. The specificationfor the novel dosage forms of this invention are indicated bycharacteristics of the active component and the particular therapeuticeffect to be achieved or the limitations inherent in the art ofcompounding such an active component for therapeutic use in warm-bloodedanimals as disclosed in this specification. Examples of suitable oraldosage forms in accord with this invention are tablets, capsules, pills,powder packets, granules, wafers, cachets, teaspoonfuls, dropperfuls,ampules, vials, segregated multiples of any of the foregoing and otherforms as herein described.

The complement inhibiting activity of a representative compound of thisinvention has been demonstrated by one or more of the followingidentified tests: (i) Test, Code 026 (C1 inhibitor). This test measuresthe ability of activated human fluid phase human C2 in the presence ofC4 and appropriate dilutions of the test compound. An active inhibitorprotects C2 from C1 and C4; (ii) Test, Code 035 (C3-C9 inhibitor). Thistest determines the ability of the late components of human complement(C3-C9) to lyse EAC 142 in the presence of appropriate dilutions of thetest compound. An active inhibitor protects EAC 142 from lysis by humanC3-C9; (iii) Test, Code 036 (C-Shunt inhibitor). In this test humanerythrocytes rendered fragile are lysed in autologous serum via theshunt pathway activated by cobra venom factor in the presence ofappropriate dilutions of the test compound. Inhibition of the shuntpathway results in failure of lysis; (iv) Forssman Vasculitis Test.Here, the well known complement dependent lesion, Forssman vasculitis,is produced in guinea pigs by intradermal injection of rabbitanti-Forssman anti-serum. The lesion is measured in terms of diameter,edema and hemorrhage and the extent to which a combined index of theseis inhibited by prior intraperitoneal injection of the test compound at200 mg/kg is then reported, unless otherwise stated; (v) Forssman ShockTest. Lethal shock is produced in guinea pigs by an i.v. injection ofanti-Forssman antiserum and the harmonic mean death time of treatedguinea pigs is compared with that of simultaneous controls; (vi)Complement Level Reduction Test. In this test, the above dosed guineapigs, or others, are bled for serum and the complement level isdetermined in undiluted serum by the capillary tube method of U.S. Pat.No. 3,876,376 and compared to undosed control guinea pigs; and (vii) Cap50 Test. Here, appropriate amounts of the test compound are added to apool of guinea serum in vitro, after which the undiluted serum capillarytube assay referred to above is run. The concentration of compoundinhibiting 50% is reported.

Table I shows that a representative compound of the invention possesscomplement inhibitory activity.

                                      TABLE I                                     __________________________________________________________________________    Biological Activities                                                                                  Assay Results                                                                 In Vitro                                               Compound               026*                                                                              035 036                                          __________________________________________________________________________    3,3'-{Ureylenebis[(6-methyl-3,1-phenylene)-                                   carbonyl]}[2,3-dihydro-2-oxonaphth[1,8-                                                                3** 2   Neg.                                         de]-1,3-oxazine-5,8-disulfonic acid], tetra-                                  sodium salt                                                                   __________________________________________________________________________       *Test identified by code herein.                                             **Numbers represent activity in wells, a serial silution assay, higher       well number indicates higher activity. The serial dilutions are two-fold.

We claim:
 1. A compound selected from those of the formula: ##STR3##wherein A is hydrogen, alkali metal or alkaline earth, with the provisothat A is identical in the same compound.
 2. A compound according toclaim 1, of the formula: ##STR4##